Human immunodeficiency virus type 1 (HIV-1) encodes for a small membrane protein known as Vpu and has two major functions in the infected cell. Vpu is known to interact with and shunt the CD4 molecule from the rough endoplasmic reticulum (RER) to the proteasome for degradation. In addition, Vpu is known to enhance virus release from infected cells. The assembly of HIV-1 viruses in CD4+ T cells lacking a vpu gene is characterized by the maturation of viruses into intracellular vesicles and the accumulation of virus particles at the cell surface. This enhanced release function of Vpu has been associated with the transmembrane domain of the Vpu molecule and investigators have shown that Vpu TM has ion channel properties (also known as a viroporin). Using pathogenic molecular clones of simian human immunodeficiency viruses (SHIV) known as SHIVKu-ibMC33, we have shown that both the transmembrane (TM) and cytoplasmic domains of Vpu protein contribute to the pathogenesis of this virus in macaques. Further, we have shown that substitution of the subtype B vpu gene from SHIVKu-ibMC33 with vpu from a subtype C HIV-1 isolate reduces the rate of CD4+ T cell loss in macaques. In the first two Specific Aims, we propose to continue our studies on the TM/ion channel of the Vpu protein in virion release. We have recently obtained a novel compound, BIT225 (from Biotron LTD.), which in our preliminary studies inhibits the release of viral particles from cultures inoculated with SHIVKu-ibwc33 but not a SHIV expressing a Vpu with a scrambled TM domain (SHIVrw)- In Specific Aim 1, we propose to examine the site/mechanism by which BIT225 inhibits SHIVKu-ibMcss replication and virus release. In the Specific Aim 2, we propose to examine the ability of these compounds to decrease replication of mutant and parental SHIVs in macaque macrophage cultures. In the third and fourth Aims, we propose to continue our studies on the biological properties of the subtype C Vpu. In Specific Aim 3, we propose to examine the role of the highly conserved dileucine motif with adaptor complexes (AP-1, AP-2, and AP-3) and to determine if this domain influences virus release from infected cells. In Specific Aim 4, we propose to generate a series of SHIVs expressing chimeric subtype B/C Vpu proteins to determine what domain is responsible for the decreased rate of CD4+ T cell loss in macaques. The results of these studies will provide novel information of the Vpu protein from the subtype C HIV-1, which accounts for the most HIV-1 infections worldwide.